Probing Chemical Complexity of Amyloid Plaques in Alzheimer’s Disease Mice using Hyperspectral Raman Imaging

One of the distinctive pathological features of Alzheimer’s disease (AD) is the deposition of amyloid plaques within the brain of affected individuals. These plaques have traditionally been investigated using labeling techniques such as immunohistochemical imaging. However, the use of labeling can disrupt the structural integrity of the molecules being analyzed. Hence, it is imperative to employ label-free imaging methods for noninvasive examination of amyloid deposits in their native form, thereby providing more relevant information pertaining to AD. This study presents compelling evidence that label-free and nondestructive confocal Raman imaging is a highly effective approach for the identification and chemical characterization of amyloid plaques within cortical regions of an arcAβ mouse model of AD. Furthermore, this investigation elucidates how the spatial correlation of Raman signals can be exploited to identify robust Raman marker bands and discern proteins and lipids from amyloid plaques. Finally, this study uncovers the existence of distinct types of amyloid plaques in the arcAβ mouse brain, exhibiting significant disparities in terms of not only shape and size but also molecular composition.

, respectively.Raman spectra are fitting using a Lorentzian function (red curve).A blue-shift of ca.7 cm -1 is observed in position of Amide I band at the location of amyloid plaques.

Figure S1 .
Figure S1.Confocal Raman spectrum of the borosilicate glass substrate used for supporting microtomed mouse brain slices in this work.Laser power: 77 mW.Acquisition time: 1 s.

Figure S2 .
Figure S2.Average Raman spectra (1633 -1713 cm -1 ) measured in the protein-rich and amyloid plaque-rich regions shown in Figures2b and 2c, respectively.Raman spectra are fitting using a Lorentzian function (red curve).A blue-shift of ca.7 cm -1 is observed in position of Amide I band at the location of amyloid plaques.

Figure S3 .
Figure S3.Optical images of the region presented in Figure 2a-d (a) before and (b) after confocal Raman imaging showing no sample damage.

Figure S4 .
Figure S4.Confocal Raman images of lipid, amyloid plaque, and proteins in a different region of the arcAβ mouse brain slice constructed using the intensities of Raman bands at (a) 2848, (b) 1660, and (c) 1667 cm -1 , respectively.(d) Overlay of the confocal Raman images shown in Panels a-c.Laser power: 25 mW.Acquisition time: 1 s.Step size: 2 μm.Amyloid plaque (green) is surrounded by a lipid-rich region (red), which is further encapsulated by a protein-rich region (blue).(e) The C-H spectral region of the average Raman spectra of the areas populated with (red trace) lipids, (green trace) plaques, and (blue trace) proteins.The Raman band at 2848 cm -1 , used to construct the image shown in Panel a is highlighted in red.(f) The amide I spectral region of the average Raman spectra of the sample areas populated with (red trace) lipids, (green trace) amyloid plaques, and (blue trace) proteins.The Raman bands at 1660 and 1667 cm -1 used to construct the images shown in Panels b and c, respectively are highlighted in blue and green.Optical images of the measured region (g) before and (h) after confocal Raman imaging showing no sample damage.Focal spot of the green excitation laser is visible in Panel g.

Figure S5 .
Figure S5.Confocal Raman images of the arcAβ mouse brain slice constructed using the intensities of all detected Raman bands in the region measured in Figure S4a-d.Laser power: 25 mW.Acquisition time: 1 s.Step size: 2 μm.Based on the spatial correlation between Raman signals, the images are classified into 6 groups.The group I signals originate from nucleic acids.The group II signals originate from lipids.The group III signals originate from proteins.The group IV signal originate from proteins.The group V signal originates from amyloid plaques.The group VI signals originate from lipids.

Figure S6 .
Figure S6.Confocal Raman images of lipid, amyloid plaque, and proteins in a different region of the arcAβ mouse brain slice constructed using the intensities of Raman bands at (a) 2848, (b) 1660, and (c) 1667 cm -1 , respectively.(d) Overlay of the confocal Raman images shown in Panels a-c.Laser power: 25 mW.Acquisition time: 1 s.Step size: 2 μm.Amyloid plaque (green) is surrounded by a lipid-rich region (red), which is further encapsulated by a protein-rich region (blue).(e) The C-H spectral region of the average Raman spectra of the areas populated with (red trace) lipids and amyloid plaque and (blue trace) proteins.The Raman band at 2848 cm -1 , used to construct the image shown in Panel a is highlighted in red.(f) The amide I spectral region of the average Raman spectra of the sample areas populated with (green trace) lipids and amyloid plaque and (blue trace) proteins.The Raman bands at 1660 and 1667 cm -1 used to construct the images shown in Panels b and c, respectively are highlighted in blue and green.Optical images of the measured region (g) before and (h) after confocal Raman imaging showing no sample damage.

Figure S7 .
Figure S7.Confocal Raman images of the arcAβ mouse brain slice constructed using the intensities of all detected Raman bands in the region measured in Figure S6a-d.Laser power: 25 mW.Acquisition time: 1 s.Step size: 2 μm.Based on the spatial correlation between Raman signals, the images are classified into 6 groups.The group I signals originate from nucleic acids.The group II signals originate from lipids.The group III signals originate from proteins.The group IV signal originate from proteins.The group V signal originates from amyloid plaques.The group VI signals originate from lipids.